This work was partially funded by the China Scholarship Council [CSC No. 201808440394]. W.C. was financed by CSC.

The protocol was reviewed and approved by the local ethics committee (approval number 287/2020B02).
1. Preparation of calcium phosphate coated cell culture plates
<ol>
	<li>Preparation of calcium stock solution (25 mM CaCl2&#183;2H2O, 1.37 M NaCl, 15 mM MgCl2&#183;6H2O in Tris buffer)
	<ol>
		<li>Prepare 50 mM Tris buffer and adjust pH to 7.4 using 1 M HCl.</li>
		<li>Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.</li>
		<li>Weigh out 0.368 g of CaCl2&#183;2H2O, 8.0 g of NaCl, and 0.305 g of MgCl2&#183;6H2O and dissolve in Tris buffer one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Store at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of phosphate stock solution (11.1 mM Na2HPO4, 42 mM NaHCO3 in Tris buffer)
	<ol>
		<li>Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.</li>
		<li>Weigh out 0.158 g of Na2HPO4&#160;and 0.353 g of NaHCO3 and dissolve in Tris buffer one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Store at room temperature.</li>
	</ol>
	</li>
	<li>Pre-calcification of 96-well cell culture plates
	<ol>
		<li>Prepare 30 mL of working solution by mixing 15 mL of 50 mM Tris buffer, 7.5 mL of calcium stock (step 1.1.4) solution, and 7.5 mL of phosphate stock solution (step 1.2.3). Filter the solution with a 0.2 &#181;m filter.</li>
		<li>Prepare a 96-well cell culture plate with flat bottom. Pipette 300 &#181;L of calcium phosphate solution (step 1.3.1.) to each well. Cover the plate with a lid and incubate the plate at 37 &#176;C for 3 days.</li>
	</ol>
	</li>
	<li>Preparation of calcium phosphate solution (2.25 mM Na2HPO4, 4 mM CaCl2&#183;2H2O, 0.14 M NaCl, 50 mM Tris base in ddH2O)
	<ol>
		<li>Add 2 mL of 1 M HCl to 40 mL of deionized water in a beaker with a magnetic stirring bead and dissolve 0.016 g of Na2HPO4, 0.0295 g of CaCl2&#183;2H2O, 0.409 g of NaCl, and 0.303 g of Tris base one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Add deionized water to fill the volume to 50 mL. Filter the solution with a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Calcification of 96-well cell culture plates
	<ol>
		<li>Aspirate pre-calcification solution from the pre-calcified 96-well cell culture plates and add 300 &#181;L of calcium phosphate solution (step 1.4.2) to each well. Cover the plates with a lid and incubate at 37 &#176;C for 1 day.</li>
		<li>Turn the plate over to pour out the solution and wash the plate three times with deionized water thoroughly. Dry the plate immediately using a hair dryer or compressed CO2 or N2 gas to maintain a uniform surface.</li>
		<li>Sterilize the coated plate by UV radiation in a clean bench for 1 h. Use the coated plate immediately or seal the plate with parafilm and store it at room temperature.</li>
	</ol>
	</li>
</ol>
2. Isolation of PBMCs from human peripheral blood
<ol>
	<li>Draw 15 mL of blood from the vein after obtaining written informed consent from the blood donor (ethical vote: 287/2020B02).</li>
	<li>Dilute 15 mL of fresh blood with an equal volume of PBS and mix by inverting the tube several times or by drawing the mixture in and out of a pipette.</li>
	<li>Prepare a 50 mL conical tube containing 15 mL of density gradient solution (e.g., Ficoll, Table of Materials) per tube. Tilt the tube and carefully layer 30 mL of diluted blood sample onto the 15 mL of density gradient solution (diluted blood sample: density gradient solution, 1:0.5-1 ratio). 
	NOTE: When overlaying the sample, pay attention not to mix the density gradient solution with the diluted blood sample.</li>
	<li>Centrifuge at 810 x g for 20 min at 20 &#176;C in a swinging-bucket rotor without brake.</li>
	<li>Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.</li>
	<li>Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.</li>
	<li>Fill the tube with PBS, mix, and centrifuge at 300 x g for 10 min at 20 &#176;C (brake can be used from this stage onwards).</li>
	<li>Carefully remove supernatant completely. Resuspend the cell pellet in 50 mL of PBS and centrifuge at 300 x g for 10 min at 20 &#176;C. Carefully remove the supernatant completely.</li>
	<li>For removal of platelets, resuspend the cell pellet in 50 mL of PBS and centrifuge at 200 x g for 10 min at 20 &#176;C. Carefully remove the supernatant completely.</li>
	<li>Transfer resuspended cells to cell culture flasks for expansion of OC progenitors.</li>
</ol>
3. Expansion of OC progenitors
<ol>
	<li>Resuspend PBMCs in complete &#945;-MEM (10% FBS, 1% Pen/Strep, 1% amphotericin B) containing 20 ng/mL M-CSF. Seed PBMCs at a density of 2.5 x 105 cells/cm2. Usually, cells isolated from 15-20 mL fresh blood can be seeded in one 75 cm2 flask (1.5-2 x 107 cells).</li>
	<li>Feed the cells with fresh complete &#945;-MEM containing 20 ng/mL M-CSF every third day until the attached cells reach a desired confluency. Typical yields are 1.5-2 x 106 cells/flask when cells are 95% confluent. Usually, the expansion period lasts 6 days. 
	&#8203;NOTE: The osteoclastogenic potential of the precursors can decrease with prolonged cultivation time.</li>
</ol>
4. Induction of osteoclastogenesis in CaP coated plates
<ol>
	<li>To facilitate cell adhesion, incubate the CaP coated plates with 50 &#181;L of FBS for 1 h in a 37 &#176;C incubator.</li>
	<li>To detach the OC precursors, wash the flask with PBS twice to remove dead or non-adherent cells. Add 4 mL of trypsin (Table of Materials) per 75 cm2 flask, for 30 min.
	<ol>
		<li>Add 4 mL of complete &#945;-MEM to stop the digestion, carefully detach the cells using a cell scraper and transfer the cells into a 50 mL tube.</li>
		<li>Determine the total cell number using a Neubauer chamber or similar.</li>
	</ol>
	</li>
	<li>Pellet the cells by centrifugation for 7 min at 350 x g. Resuspend the pellet in sufficient complete &#945;-MEM containing 20 ng/mL M-CSF and 20 ng/mL RANKL to get a concentration of 1 x 106 cells/mL.</li>
	<li>Aspirate FBS solution from the CaP coated 96-well plate and pipette 200 &#181;L of cell suspension per well (2 x 105 cells/well).</li>
	<li>Incubate the OC precursors for the desired time period or with the desired reagents relevant to the experimental design. Usually, high numbers of large and multinucleated OCs can be observed after 6 days and when resorption pits are forming. Feed cells with fresh complete &#945;-MEM medium containing 20 ng/mL M-CSF and 20 ng/mL RANKL every third day.</li>
	<li>At the end of incubation, wash cells with PBS twice, fix with 4% paraformaldehyde for 10 min, and wash with PBS again.</li>
	<li>Use the fixed OCs directly for fluorescence staining or store at 4 &#176;C.</li>
</ol>
5. Fluorescence staining of OCs and CaP coating
<ol>
	<li>Incubate the fixed cells with permeabilization buffer (0.1% Triton in PBS) for 5 min.</li>
	<li>Stain actin filaments with 100 &#181;L of AlexaFluor 546 labeled phalloidin solution in PBS for 30 min and aspirate the staining solution. Add 100 &#181;L of Hoechst 33342 staining solution (10 &#181;g/mL in PBS) for 10 min to stain nuclei. DAPI can also be used.</li>
	<li>Stain CaP coating with 100 &#181;L of 10 &#181;M calcein in PBS for 10 min. Wash three times with PBS and take images.</li>
	<li>After fluorescence imaging the same plates can be used to quantify resorption pit area by Von Kossa staining. To do so, wash the plates with deionized water twice.</li>
</ol>
6. Quantification of total resorption pit area
<ol>
	<li>To stain CaP coating with Von Kossa staining, incubate with 50 &#181;L of 5% AgNO3 in deionized water per well under UV radiation for 1 h, until the coating on the bottom of the wells has turned brown.</li>
	<li>Wash the plate three times with deionized water and take brightfield images. An objective with little magnification, such as a 1.25x objective, can be used to capture the whole well in one image. This facilitates subsequent image analysis. Alternative methods to capture the whole area of the well can be applied.</li>
	<li>Open an image file with ImageJ: File | Open | Image | Type | 8-bit. Verify the scale unit in the lower right corner of the image using Straight Line | Analyze | Set Scale. Enter the length in the &#34;known distance&#34; blanket, enter new scale unit in Unit of length, and check the box Global.</li>
	<li>List measurement parameters to be analyzed: Analyze | Set Measurements. In the Set Measurements window, check the Area and Limit to threshold boxes.</li>
	<li>Measure the area of pits: Image | Adjust | Threshold. In the Threshold window, check box Dark background and click Auto. The area of pits turns red. Close the Threshold window and select Analyze | Measure. Save the result file: File | Save As.</li>
</ol>
7. Quantification of OC number and size, and normalized resorption pit area
<ol>
	<li>Open a fluorescence image of osteoclasts with ImageJ and verify the scale unit (see step 6.3).</li>
	<li>List measurement parameters to be analyzed: Analyze | Set Measurements. In the Set Measurements window, check the Area box.</li>
	<li>Outline the OCs using ROI Manager and Polygon selections: Analyze | Tools | ROI Manager. Click Polygon selections in toolset, outline one OC (cells with actin ring and &#8805; three nuclei) and click Add [t] in ROI Manager window. Repeat outlining until all OCs are included.</li>
	<li>Measure number and size of OCs: Select all items in ROI Manager and click Measure. Save the result file: File | Save As (Figure 3E).</li>
	<li>Measure the pit area of fluorescence images. Open the fluorescent image of coating correlated with the image in step 7.3 and verify the scale unit (see step 6.3).
	<ol>
		<li>List measurement parameters to be analyzed (see step 6.4). Select Image | Adjust | Threshold. In the Threshold window, uncheck all boxes and click Auto. The area of the pits turns red. Close the Threshold window and select Analyze | Measure. Save the result file.</li>
	</ol>
	</li>
	<li>Calculate normalized pit area by OC number.</li>
</ol>

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The authors have nothing to disclose.

Here we describe a simple and reliable method for an osteoclastic resorption assay using OCs derived and expanded in vitro from PBMCs. The used CaP coated cell culture plates can be easily prepared and visualized using lab-available materials. In addition to unsorted PBMCs adopted in this protocol, OCs generated from murine monocytic cells21 and bone marrow macrophage cells17 have also been cultured on similar synthetic substrates for pit assay, thus these cell sources can be moved to this approach referring to the corresponding literature.
For this protocol, we used a simple in-lab fabricated CaP coating instead of expensive commercially available assay plates. Although bone and dentin slices are two common and affordable resorbable materials for resorption pit assay, the availability and complicated preparation are major drawbacks. As an easily prepared synthetic substrate, CaP coating is more convenient and readily available than the two original materials. Another advantage of this pit assay approach is that cells can be visualized under a bright field microscope during culture, whereas cells growing on opaque bone and dentin slices cannot be observed.
This CaP coating method can be flexibly adapted according to the required experimental needs to different cell culture plate sizes.
Unlike previously reported17,21, we incubate the plates with coating solution at 37 &#176;C instead of at room temperature, resulting in the spontaneous growth of apatite nuclei on cell culture plates at body temperature. CaP coated plates are available for experiments immediately after being dried and sterilized by UV radiation, which saves time compared to previously reported methods17,21.
Filtration of the coating solution (Step 1.3.1 and 1.4.2) is a crucial step, which helps to obtain a uniform particle size and reduce uncoated spots within the coating caused by air bubbles and impurities in the solution. Drying the plate immediately (step 1.5.2) is another critical step, which helps to make a uniform CaP coating. Otherwise, it is prone to form a thicker coating in the middle of the well.
It is worth pointing out that the thickness of the coating and the density of calcium phosphate deposits depend on the volume of coating solution within a certain range. Therefore, it is possible to control coating thickness by changing the volume of coating solution in the well. However, it is suggested to add as much volume of coating solution as possible when the 96-well cell culture plate is used because of the small total volume for this culture plate format. The inner wall of the well is also coated by CaP during both coating steps, therefore caution is required to not touch the bottom nor the wall with the pipette in order to protect the coating from damage.
According to our observation, most of OC precursors expanded from PBMCs are able to attach to the CaP coating without any problems. However, soaking the coated plate with FBS before cell seeding improves the cell adhesion efficiency, as previously reported21.
Detaching the OC precursors gently using a cell scraper (step 4.2) is also important because most of the OC precursors were still adherent, and minimizing the mechanical injury is favorable for cell viability.
A limitation of this method is that the bulk PBMCs that we used as a source of OC precursors is a highly mixed cellular source, of which only a small fraction of cells (CD14+ cells) are actual OC precursors. Since other cells capable of affecting osteoclastogenesis (such as stromal cells and lymphocytes) are present amongst isolated PBMCs, this may impact assay results in ways that may be hard to predict, and might confound assay interpretation. To better investigate direct effects of growth factors, cytokines or glucocorticoids on resorptive activity of OCs, methods of OC precursor purification (e.g., fluorescence-activated cell sorting (FACS) and magnetic beads purification20 based on specific cell-surface markers23,24,25,26) are recommended.
The limitations of the CaP substrate in obtaining brightfield images of both cells and underlying pits for the same area is caused by the incompatibility of TRAP and Von Kossa staining, so that normalized resorption area of the entire well is not available. But instead, fluorescence imaging can be used to visualize resorption area and cell number to obtain normalized resorption data. These data may provide important insight into whether the increase in total resorption area under experimental conditions is due to increased osteoclast number alone or increased resorption capacity of individual cells. In addition, it allows the study of the relative effects of osteoclast number, size, nucleus number, and resorption capacity.
In summary, we describe a useful and simple protocol for a resorption pit assay using a two-step calcium phosphate coating and OCs derived from primary human PBMCs. This protocol provides an easy method to establish an in vitro bone resorption model for studies related to osteoclastic resorption, and could be applied to study treatments for bone disorder diseases.

<a href="https://app-jove-com.bdigitaluss.remotexs.co/t/6601">This corrects</a> the article <a href="https://dx-doi-org.bdigitaluss.remotexs.co/10.3791/64016">10.3791/64016</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro</a>. The Protocol section was&#160;updated.

Erratum: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Osteoclasts (OCs) are tissue-specific macrophages derived from hematopoietic stem cells (HSCs), playing a pivotal role in bone remodeling together with osteoblasts1. Sex hormone-induced, immunological, and malignant bone disorders that destroy bone systemically or locally are due to excess osteoclastic activity, including menopause-related osteoporosis2, rheumatoid arthritis3, periodontal disease4, myeloma bone disease5, and osteolytic bone metastasis6. In contrast, defects in OC formation and function can also cause osteopetrosis7. HSCs undergo differentiation into OC progenitors under macrophage colony-stimulating factor (M-CSF, gene symbol ACP5) stimulation. In the presence of both M-CSF and receptor activator of NF-&#954;B ligand (RANKL, gene symbol TNFSF11), OC progenitors differentiate further into mononuclear OCs and subsequently fuse to become multinucleated OCs8,9,10. Both cytokines M-CSF and RANKL are indispensable and sufficient for induction of osteoclastic markers such as calcitonin receptor (CT), receptor activator of nuclear factor &#954; B (RANK), proton pump V-ATPase, chloride channel 7 alpha subunit (CIC-7), integrin &#946;3, tartrate-resistant acid phosphatase (TRAP, gene symbol ACP5), lysosomal cysteine protease cathepsin K (CTSK), and matrix metallopeptidase 9 (MMP9). Activated OCs form a sealing zone on the bone surface through the formation of an actin ring with a ruffled border11,12. Within the sealing zone, OCs mediate resorption through secreting protons via the proton pump V-ATPase12,13, MMP914, and CTSK15, leading to the formation of lacunae.
For in vitro experiments, OC progenitors can be obtained by expansion of bone marrow macrophages from mice&#39;s femur and tibia16,17, as well as by isolation of human peripheral blood mononuclear cells (PBMCs) from blood samples and buffy coats18,19,20, or by differentiation of the immortalized murine monocytic cells RAW 264.721,22.
In the present protocol, we describe an osteoclastic resorption assay in CaP coated cell culture plates using OCs derived from primary PBMCs. The CaP coated cell culture plates method used here are adopted and refined from the method described previously by Patntirapong et al.17 and Maria et al.21. To obtain OC precursors, PBMCs are isolated by density gradient centrifugation and expanded as described previously20.

<table><tbody><tr><td>AgNO&lt;sub&gt;3&lt;/sub&gt;</td><td>SERVA Electrophoresis GmbH</td><td>35110</td><td>Silver nitrate</td></tr><tr><td>a-MEM</td><td>Gibco</td><td>32561-029</td><td>MEM alpha, GlutaMAX, no nucleosides</td></tr><tr><td>amphotericin B</td><td>Biochrom</td><td>03-028-1B</td><td>Amphotericin B Solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>21097-50G</td><td>Calcium chloride Dihydrate</td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td>Calcein</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F7524</td><td>fetal bovine serum</td></tr><tr><td>Ficoll</td><td>Cytiva</td><td>17144002</td><td>Ficoll Paque Plus</td></tr><tr><td>Fixation buffer</td><td>Biolegend</td><td>420801</td><td>Paraformaldehyde</td></tr><tr><td>HCl</td><td>Merk</td><td>1.09057.1000</td><td>Hydrochloric acid</td></tr><tr><td>Hoechst 33342</td><td>Promokine</td><td>PK-CA707-40046</td><td>Hoechst 33342</td></tr><tr><td>M-CSF</td><td>PeproTech</td><td>300-25</td><td>Recombinant Human M-CSF</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7791-18-6</td><td>Magnesium chloride</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem GmbH</td><td>A2943,0250</td><td>di- Sodium hydrogen phosphate anhydrous</td></tr><tr><td>NaCl</td><td>Merk</td><td>S7653-250G</td><td>Sodium chloride</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merk</td><td>K15322429</td><td>Bicarbonate of Soda</td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td>Dulbecco&#039;s Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium</td></tr><tr><td>Pen-Strep</td><td>Lonza</td><td>DE17-602E</td><td>Penicillin-Streptomycin Mixture</td></tr><tr><td>Phalloidin-Alexa Fluor 546</td><td>Invitrogen</td><td>A22283</td><td>Alexa Fluor 546 Phalloidin</td></tr><tr><td>RANKL</td><td>PeproTech</td><td>310-01</td><td>Recombinant Human sRANK Ligand (E.coli derived)</td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>93362</td><td>Tris(hydroxymethyl)aminomethan</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Alkyl Phenyl Polyethylene Glycol</td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605010</td><td>Recombinant cell-dissociation enzymes</td></tr></tbody></table>

a simple pit assay protocol to visualize and quantify osteoclastic resorption in vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

The calcium phosphate coating on the bottom of cell culture plates was performed in two coating steps comprising a 3-day pre-calcification and a 1-day calcification step. As shown in Figure 1, uniformly distributed calcium phosphate was obtained on the bottom of the 96-well plates. The coating adhered very well to the bottom after the performed washing steps.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64016/64016fig01.jpg" /> 
Figure 1: Representative brightfield image of the calcium phosphate coating on 96-well cell culture plates. The coating was carried out in two coating steps comprising a 3-day pre-calcification step and a 1-day calcification step. The scale bar represents 200 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64016/64016fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
OC precursors derived from human PBMCs were cultured on the CaP coated plates. Resorption pits and large OCs (blank area) were formed in the presence of M-CFS and RANKL after 9 days of culture (Figure 2A). The multinucleated OCs located in the pits of the CaP coating expressed high levels of TRAP (Figure 2B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64016/64016fig02.jpg" /> 
Figure 2: TRAP expression by OCs after maturation on the CaP coated cell culture plates. OC precursors were cultured on the CaP coated plates in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL for 9 days. (A) Blank area represented resorption pits. (B) Several multinucleated TRAP-positive (purple) OCs were observed within the resorption pits. The scale bar represents 200 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64016/64016fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To further characterize the maturation of OCs on the CaP coated plates, cells were stained for actin (red fluorescence). Cell nuclei were stained with Hoechst (blue) and calcein was used for calcium visualization in green. Resorption pits are visible as black areas in the green CaP coating (Figure 3B). Functional mature OCs showed three or more nuclei and the characteristic actin ring which is essential for the osteoclastogenic resorption (Figure 3A,C). The pit area of fluorescence images can be measured using the thresholding tool of ImageJ (Figure 3D). The number and size of OCs can be calculated using the ROI Manager and Polygon selection tool of ImageJ (Figure 3E).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64016/64016fig03.jpg" /> 
Figure 3: Morphology of mature OCs on the CaP coating. OC precursors were cultured on the CaP coated plates for 9 days in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL. (A) OCs were stained for actin and nuclei by phalloidin-Alexa Fluor 546 and Hoechst 33342. (B) CaP coating was stained by calcein. Black areas represent resorption pits. (C) Merged image. (D) Quantification of pit area (red area) using the thresholding tool of ImageJ. (E) OC outlining and counting using the ROI Manager and Polygon selection tool of ImageJ. The scale bar represents 500 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64016/64016fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the quantification of the resorption pits on the CaP coated cell culture plates, calcium was stained by incubation with 5% AgNO3 (Von Kossa staining). As shown in Figure 4A, OC precursors did not reach full functionality in the absence of RANKL and were not able to form resorption pits. Osteoclastogenic pits were observed only in the presence of both factors M-CSF and RANKL (Figure 4B). The resorption pit area was quantified with the thresholding tool of ImageJ (Figure 4C).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64016/64016fig04.jpg" /> 
Figure 4: Visualization and quantification of resorption pits. OC precursors were cultured on the CaP coated 96-well cell culture plates in the absence of RANKL (A) and in the presence of both factors M-CSF and RANKL (B). (C) Numbers of formed resorption pits were quantified using the thresholding tool of ImageJ. Scale bar represents 1,000 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64016/64016fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro at JoVE.com

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

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5.9K Views.  University Hospital Tübingen. Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Video: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

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Developmental Biology

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

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Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Immunology and Infection

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Biology

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Tartrate-Resistant Acid Phosphatase Staining: An In Vitro Technique to Detect TRAP Enzyme-Containing Cultured Osteoclasts

Source: Dai, Q., et al. A <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56468">RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation.</a> J. Vis. Exp. (2018).
This video demonstrates the tartrate-resistant acid phosphatase staining technique to detect the presence of TRAP-containing granules inside osteoclasts.&#160; The technique forms magenta-colored dye granule deposits in the cytoplasm of osteoclasts, rendering them visible under an inverted microscope.

Source: Dai, Q., et al. A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation. J. Vis. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Biomolecule staining

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Source: Cen, W., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.</a> J. Vis. Exp. (2022)
In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.

Source: Cen, W., et al. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (2022)
In this video, we ...

Assay Techniques

Cell-based assays

In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

Source: Kampleitner, C. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58077">Biological Compatibility Profile on Biomaterials for Bone Regeneration.</a> J. Vis. Exp. (2018)
In this video, we describe a method for the detection of osteoblast-secreted calcium deposits using an anionic dye, Alizarin Red S. The cultured osteoblasts are first stained and then the dye is recovered for colorimetric quantification of calcium deposits or the degree of osteoblast mineralization.

Source: Kampleitner, C. et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018)
In this video, we describe a ...

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Summary

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Chapters in this video

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14⁺ Monocytes

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Osteoclast Derivation from Mouse Bone Marrow

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Tartrate-Resistant Acid Phosphatase Staining: An In Vitro Technique to Detect TRAP Enzyme-Containing Cultured Osteoclasts

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction